The objective of the proposed research is to deduce mechanistic and regulatory principles that control the activities of sirtuin enzymes in yeast and human cells. These principles will be used to design small molecules that can activate sirtuins in cells. Sirtuins regulate a host of biologically significant activities including stress resistance, gene silencing and longevity. Sirtuins are implicated in mediating biological effects associated with calorie restriction. Calorie restriction has been shown to have numerous positive health benefits in mammals including reduced adipogenesis, insulin sensitivity and increased lifespan. These biological effects have raised interest in the enzymatic mechanisms of sirtuins, the means by which they are regulated in cells and the ways in which they might be modulated pharmacologically for improved human health. Sirtuins are NAD dependent deacetylases that remove acetyl-groups of acetyllysine modified histones and transcription factors thereby regulating chromatin and gene expression. We and others have demonstrated that these actvitities are regulated by NAD and nicotinamide levels in cells, and that the sirtuins are able to integrate information from energy and metabolic states to control genetic events. As part of our goal to better understand the functions of these enzymes and how they can be modulated in cells we propose to investigate the following specific aims: In Aim1 we propose to characterize the biochemical functions of this unique class of enzymes, emphasizing their chemical novelty and the incorporation of NAD in deacetylation reactions. In Aim2 we propose to show how enzymatic activity provides a mechanism for nicotinamide regulation of sirtuin activity. In addition with a recently developed mass spectrometry method we hope to gain new insights into how nicotinamide regulates sirtuins in cells. In Aim 3 we explore the development of small molecule activators of sirtuins designed from knowledge of the sirtuin reaction mechanism and the mechanism of nicotinamide regulation. These activators embody a novel approach to upregulate sirtuin action in cells and provide a potential entrypoint for pharmacological intervention to increase cell stress resistance and cell survival. Achievement of these aims is expected to provide new insights into the biochemistry and regulation of these enzymes, and provide proof of concept for new therapeutics that can activate sirtuins to treat diabetes and degenerative disorders.